Keywords

Prostasin, EGFR, prostate cancer, SREBP, Slug, Matriptase

Abstract

The glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin (PRSS8) is expressed at the apical membrane surface of epithelial cells and acts as a suppressor of tumor invasion when re-expressed in highly invasive human prostate and breast cancer cell lines. To better understand the molecular mechanisms underlying the anti-invasion phenotype associated with prostasin re-expression in prostate cancer cells, we expressed wild-type human prostasin or a serine active-site mutant prostasin in the PC-3 human prostate carcinoma cells. Molecular changes were measured at the mRNA and the protein levels. The expression of several invasion-promoting molecules is regulated by prostasin re-expression, mediated by a protein-level down-regulation of the epidermal growth factor receptor (EGFR). As a result, the cellular response to EGF was reduced as shown by the down-regulation of EGF-stimulated Erk1/2 phosphorylation. The expression of Slug, urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and granulocyte-macrophage colony stimulating factor (GM-CSF) was also down-regulated by prostasin re-expression in the PC-3 cells. Co-expression of prostasin and its activating protease matriptase with EGFR in FT-293 cells induces an apparent proteolytic cleavage of the EGFR in the extracellular domain at two specific sites, generating two N-terminally truncated EGFR fragments, named EGFR135 and EGFR110. The EGFR110 is constitutively tyrosine-phosphorylated, and in its presence the phosphorylation of downstream signaling molecules including Erk1/2 and Akt is increased under serum-free conditions. Neither EGFR135 nor EGFR110 is responsive to EGF stimulation. Deletions of the EGFR extracellular domain (ECD) were generated to map the matriptase-prostasin cleavage sites. Two candidate sites were localized to regions AA1-273 and AA273-410. These data support a mechanism of action for the matriptase-prostasin epithelial extracellular serine protease activation cascade by proteolytically modulating the EGF-EGFR signaling. Prostasin gene expression is down-regulated in high-grade and hormone-refractory prostate cancers. We investigated the mechanisms by which androgens regulate prostasin expression in the prostate and prostate cancer. We treated the LNCaP human prostate cancer cells with dihydrotestosterone (DHT) and measured the mRNA expression of prostasin and potential transcription regulators of prostasin predicted by interrogation of the prostasin gene promoter sequence. Prostasin mRNA expression in the LNCaP cells was not responsive to DHT treatment. DHT marginally up-regulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of a prostasin promoter-reporter and SREBP cDNA in HEK-293 cells resulted in stimulation of the promoter activity at ~2 fold by SREBP-1c, and up to 6 fold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of the promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2 s stimulation of the prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 cells resulted in up-regulation of the endogenously expressed prostasin while transfection of a SLUG cDNA in the LNCaP cells repressed prostasin expression. Multiple SREBP-2 binding sites, known as sterol regulatory elements (SRE s), were identified at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Mutagenesis of the five SRE s was carried out to evaluate their roles in SREBP-2 up-regulation of prostasin. SRE+98, a novel functional sterol regulatory element was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. CONCLUSIONS: Prostasin regulates the expression of several invasion-promoting molecules in prostate cancer cells by down-modulating the EGF-EGFR signaling pathway. Active prostasin induces proteolytic cleavage in the EGFR ECD at two specific sites. One of the N-terminally truncated EGFR, the EGFR110 is auto-phosphorylated along with increased phosphorylation of downstream signaling molecules. The effect of the androgen DHT on prostasin expression in prostate cells is mediated via SREBP s, which stimulate the promoter, and Slug, which represses the promoter. Slug is up-regulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

Notes

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Graduation Date

2007

Semester

Summer

Advisor

Chai, Karl

Degree

Doctor of Philosophy (Ph.D.)

College

Burnett College of Biomedical Sciences

Department

Biomolecular Science

Degree Program

Biomolecular Sciences

Format

application/pdf

Identifier

CFE0001782

URL

http://purl.fcla.edu/fcla/etd/CFE0001782

Language

English

Release Date

July 2008

Length of Campus-only Access

None

Access Status

Doctoral Dissertation (Open Access)

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